ErbB-2 is amplified and overexpressed in 20-30% of breast tumors. Today, ErbB-2 positive breast cancers are treated with trastuzumab plus chemotherapy. In spite of dramatic improvements in survival for women with ErbB-2+ breast tumors, resistance remains a major problem in metastatic disease. We have recently published that ErbB-2 suppresses Notch-1 activity and this is reversed by trastuzumab or a dual EGFR/ErbB-2 tyrosine kinase inhibitor similar to lapatinb. Furthermore, we demonstrated that trastuzumab resistance is reversed with a Notch-1 siRNA or a gamma-secretase inhibitor (GSI). Coincidentally, co-overexpression of Notch-1 and its ligand, Jagged-1, in breast cancer have been associated with the poorest overall survival. We know that activated Notch-1 plays an important role in ErbB-2 positive breast cancer and trastuzumab resistance; however, the precise mechanism by which Notch-1 is activated is not yet known. Thus, we hypothesized that overexpression of ErbB-2 inhibited expression and/or cell surface localization of Jagged-1 to suppress Notch-1 activity. This would be reversed in response to anti-ErbB-2 treatment. The results demonstrated that trastuzumab or lapatinib treatment of SKBr3 cells increased protein expression of Jagged-1 by Flow Cytometry. Notch-1 and Jagged-1 co-localized with early endosome antigen-1 (EEA-1) to submembraneous endosomes in ErbB-2 positive breast cancer cells as measured by confocal immunofluorescence. However upon trastuzumab treatment, Notch-1 and Jagged-1 exited EEA-1 positive vesicles and Notch-1 accumulated in the nucleus while Jagged-1 localized to the cell membrane. Cell surface biotinylation assays showed that Jagged-1 was enriched on the cell surface in response to trastuzumab treatment and this was mimicked by expression of K44A Dynamin. Cell surface accumulation of Jagged-1 was associated with an increase in Notch-1 transcriptional activity as measured by an increase in Deltex1 mRNA by real-time PCR. Conversely, knockdown of Jagged-1 by siRNA in SKbr3 and BT474 cells prevented the trastuzumab-induced increase in Deltex1 mRNA. Co-culture studies confirmed that culturing SKBr3 cells with fibroblast-expressing Jagged-1 or DLL4 increased Notch activity as measured by a CBF-1/Notch reporter assay. Taken together, these results suggest that ErbB-2 suppresses Notch-1 activity by retention of Jagged-1 or possibly other Notch ligands within early endosomes, which may be reversed by trastuzumab or lapatinib treatment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 614.